Cell-cell fusion mediated by HAP2/GCS1 identifies it as a gamete fusion protein. While any form of sexual reproduction depends on cell-cell fusion, the machinery mediating sperm-egg fusion is yet to be identified. While HAP2/GCS1 proteins have been implicated as potential gamete fusogens in Arabidopsis, Chlamydomonas, Tetrahymena, Dictyostelium and Plasmodium, it remained unknown whether this protein directly mediates gamete fusion, acts as an accessory molecule that regulates activity of yet-unidentified real protein fusogen or is involved in pre-fusion stages such as signaling or tight adhesion. In our recent study (1), collaboration with Drs. Benjamin Podbilewiczs and Pablo Aguilars laboratories, we show that Arabidopsis HAP2/GCS1 expression in mammalian cells is sufficient to promote cell-cell hemifusion and fusion. Intriguingly, structural modeling of the HAP2/GCS1 protein family suggests striking similarity between HAP2/GCS1 and C. elegans fusogens EFF-1 and AFF-1. Unexpectedly, we found that HAP2/GCS1-mediated hemifusion and fusion requires the presence of the protein in both fusing membranes. This is the first evidence that a homotypic fusogen has to be present in both membranes to mediate even hemifusion, a fusion stage that is less energy intensive than opening and expansion of a fusion pore (2). This bilateral requirement can be bypassed by replacing the plant gene with C. elegans EFF-1 somatic cell fusogen in one of the fusing cells or the virus, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Within a month after our paper, two more independent studies (3,4), including X-ray crystallographic demonstration of striking similarity between Chlamydomonas HAP2/GCS1, fusion proteins of dengue and Zika viruses, and EFF-1, have further substantiated the conclusion that HAP2/GCS1 is indeed a protein fusogen. Still, so far, our work remains the only study that presents functional evidence for this conclusion. Future work is needed to clarify the molecular mechanisms by which the first gamete fusogen HAP2/GCS1 mediates fusion. Fusion stage of HIV-1 entry depends on virus-induced cell surface exposure of phosphatidylserine. Human Immunodeficiency virus 1 (HIV-1), the causative agent of AIDS, delivers its RNA into cells by fusing the viral envelope with the cell membrane. This fusion process is mediated by viral envelope glycoprotein Env, a trimer of heterodimers consisting of gp120 and gp41 subunits. Fusion is initiated by gp120 interactions with CD4 and one of the two coreceptors CCR5 and CXCR4 at the surfaces of the target cells. A number of studies and, especially, studies of resting primary cells, have suggested that an efficient Env-mediated fusion and infection also depends on intracellular signaling. Specifically, Ca2+ signaling is triggered by engagement of the coreceptors with gp120 (5-9). However, the role of signaling in HIV-1 fusion/infection remains controversial and appears to be cell type- and activation status-dependent. In our recent study (10) (collaboration with Drs. Leonid Margolis and Gregory Melikian laboratories), we report that HIV-1 binding to its receptors induces non-apoptotic exposure of PS at the surface of the target cell and that externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Specific interactions between the gp120 subunit of Env of cell-surface-bound virions and coreceptors triggered Ca2+ signaling-dependent TMEM16F-mediated PS externalization in the plasma membrane. Blocking externalized PS with PS-binding proteins or suppressing TMEM16F function inhibited Env-mediated fusion at a stage that follows the formation of pre-fusion Env-CD4-coreceptor complexes and precedes the gp41 restructuring, which brings about hemifusion. Exogenous PS added to the plasma membrane promoted fusion and the extent of this promotion of HIV fusion increased for the target cells with lower levels of coreceptor expression and upon reduction of the number of fusion-competent Envs. We also found both single round infection with HIV Env pseudoviruses and replicative infection with live virus, including HIV-1 infection of human lymphoid tissue ex vivo, to depend on PS externalization in target cells. This dependence is conserved between X4-tropic viruses and R5-viruses, including physiologically relevant high CD4-requiring, non-macrophage-tropic R5 virus JR-CSF. Our findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion Env-CD4-coreceptor complexes. A similar promotion of Env-mediated fusion by PS and another anionic lipid phosphatidylglycerol suggests the importance of electrostatic interactions rather than specific interactions with the polar head group of PS. We propose that PS at the surface of the target cell lowers the minimal number of coreceptor molecules that need to be engaged by each Env trimer to initiate gp41 refolding. The negatively charged PS on the target membrane can draw out the positively charged regions of a coreceptor-free gp120 monomer such as V3 loop that are exposed after gp120-CD4 binding (11). These electrostatic interactions may stabilize intermediate conformations of gp120 and facilitate gp41 release from the gp120 grip. An especially strong fusion promotion by exogenous PS for the target cells with relatively low density of accessible CCR5 may reflect a stronger dependence of their fusion on gp120 trimer-coreceptor complexes with fewer than three engaged coreceptors. However, we found that fusogenic restructuring of Env depends on cell-surface PS even for target cells with exceptionally high levels of CCR5 expression such as TZM-bl cells. A very strong dependence of HIV-1 fusion on surface PS and, by extension, on the signaling triggered by Env-coreceptor interactions observed for the JC10 cells with low co-receptor densities, approaching those characteristic for resting peripheral blood lymphocytes, is consistent with the hypothesis that signal transduction is essential in physiologically relevant conditions (reviewed in (8). The uncovered link between HIV-1 infection and PS externalization identifies a bi-directional signaling pathway in which the classic outside-in signaling through GPCR-coreceptor triggers, via intracellular Ca2+ rise, inside-out PS externalization signaling mediated by TMEM16F. In the context of HIV entry, our findings suggest that within the diverse populations of target cells HIV-1 infects the CD4- and coreceptor-expressing cells that mount the signaling responses that support viral entry and infection. Since disrupting the PS externalization pathway suppressed HIV-1 infection, this pathway may present new targets for development of anti HIV-1 drugs. Finally, PS dependence that we uncovered here for HIV-1 entry can be shared by some other viruses (see (10) for references). References: 1. Valansi, C. et al., (2017) J Cell Biol 216, 571-581 2. Chernomordik, L. V., and Kozlov, M. M. (2005) Cell 123, 375-382 3. Fedry, J. et al., (2017) Cell 168, 904-915 e910 4. Pinello, J. F. et al., (2017) Curr Biol 27, 651-660 5. Davis, C. et al, (1997) J Exp Med. 186, 17931798 6. Harmon, B., and Ratner, L. (2008) J. Virol. 82, 9191-9205 7. Wu, Y., and Yoder, A. (2009) PLoS pathogens 5, e1000520 8. Wilen, C. B. et al. (2012) Cold Spring Harb Perspect Med 2 9. Melar, M. et al. (2007) J Virol 81, 1773-1785 10. Zaitseva, E. et al., (2017) Cell Host Microbe 22, 99-110 e117 11. Kwong, P. D. et al., (2000) J Virol 74, 1961-1972